The laboratory rat is the preferred rodent model in pre-clinical drug studies and encompasses the bulk of accumulated knowledge in drug development. Their larger size facilitates procedures otherwise difficult in mice, including studies using instrumentation, blood sampling, and surgeries. Although rats are more suitable than mice for pharmacological, toxicological, physiological, and many biological assays, the absence of genetic engineering technologies suitable for use in the rat has made the mouse the preeminent rodent model. However, the recent emergence of new and more precise gene targeting techniques for the rat has resulted in significant growth in the production of genetically modified rats, which will undoubtedly expand at an exponential rate. A limiting factor in the adoption of genetically modified rats as the rodent model of choice has been the expensive, inefficient, and labor intensive manipulations required for their production, resulting in costs as high as $50,000 to $100,000 per genetically modified rat line. Consequently, it is essential to develop more viable technologies for rat genome engineering that result in more accurate, efficient, and cost-effective production of genetically modified rats. Transposagen Biopharmaceuticals, Inc. is the worldwide leader in the generation of genetically modified rat models and is uniquely positioned to facilitate advancements in this field. Precision BioSciences develops custom genome engineering enzymes based on engineered homing endonucleases. This research proposal is a collaborative effort to merge the two technologies and test the feasibility of using engineered homing endonucleases as an extremely specific and efficient method to produce genetically modified rats. In Phase I we will attempt to knockout the rat Rag1 gene in spermatagonial stem cells (SSCs) using a Rag1-specific endonuclease. These modified cells will then be used to generate knockout Rag1 mutant rats. For comparison, we will attempt to achieve the same result using conventional gene targeting methods in SSCs. The incorporation of an engineered endonuclease in SSCs should greatly streamlines the knockout rat production process, Transposagen Biopharmaceuticals and Precision BioSciences will collaborate in a large-scale Phase II project to generate precisely engineered high-value rat models. Success in this project will significantly reduce the cost of transgenic rat production and give academic and industry investigators greater access to improved rodent models. PUBLIC HEALTH RELEVANCE: The laboratory rat has been a valuable animal model for biomedical research due to its similarity to human physiology. However, the ease and lower costs associated with generating mutations in mice has lead to a greater reliance on genetically engineered mouse models despite the inability of many of these models to mimic human diseases. We outline a novel strategy that integrates our expertise in spermatogonial stem cells (SSCs) with a site-specific enzyme technology to increase efficiency of mutagenesis. We aim to establish an innovative transgenic rat service that will provide clients with a lower cost and more efficient alternative to the current genetically modified rat technology. Thus, if feasibility is demonstrated, this project would benefit many goals of public health by making the production of mutations in the rat that model human diseases readily accessible to the research community.